Identification And Functional Dissection Of Rel_ANA,a Gene Invovled In Stringent Response In Anabaena SP. PCC7120

Anabaena sp. PCC7120(Anabaena) is a filamentous cyanobacterium capable of nitrogen fixation. When deprived of combined-nitrogen source in the growth medium, the filamentous cyanobacterium Anabaena can form heterocysts capable of nitrogen fixation. Heterocysts are semi-regularly spaced among vegetative cells, which accunt for10%-20%of the cells along each filament. Heterocysts fix nitrogen and provide nitrogen source to vegetative cells. The process of heterocyst differentiation takes about20-24h, during which extensive metabolic and morphological changes take place.Guanosine tetraphosphate (ppGpp) is the signal of the stringent response that ensures cell survival by adjusting major cellular activities in response to nutrient starvation in bacteria, and ppGpp accumulates at the early stage of heterocyst differentiation. Here we show that all1549(designated as relana) in Anabaena, homologous to relA/spoT, is upregulated in response to nitrogen deprivation and predominantly localized in vegetative cells. The complementation test in Escherichia coli showed that Relana possesses ppGpp synthase and hydrolase activity. The disruption of relana strongly affects the synthesis of ppGpp, and the resulting mutant, MHRS, fails to form heterocysts and to grow in the absence of a combined-nitrogen source. This phenotype can be complemented by a wild type copy of relana-Although the upregulation of hetR is affected in the mutant, ectopic overexpression of hetR cannot rescue the phenotype. However, we found that the mutant rapidly loses its viability, within a time window of3-6h, following the deprivation of combined nitrogen. We propose that ppGpp plays a major role in rebalancing the metabolic activities of the cells in the absence of nitrogen source supply, and this regulation is necessary for filament survival and consequently the success of heterocyst differentiation.